Exopolysaccharides from Klebsiella oxytoca: anti-inflammatory activity

Abstract Exopolysaccharides (EPS) produced by Klebsiella oxytoca are of environmental, pharmaceutical, and medicinal interest. However, studies about the anti-inflammatory activity of EPS produced by this microorganism still remain limited. The aim of this study was to produce, characterize, and evaluate the anti-inflammatory activity of EPS from K. oxytoca in a pleurisy model. Colorimetric analysis revealed that precipitated crude exopolysaccharides (KEPSC) and deproteinated exopolysaccharides (KEPS) present high levels of total carbohydrates (65.57% and 62.82%, respectively). Analyses of uronic acid (7.90% in KEPSC and 6.21% in KEPS) and pyruvic acid (3.01% in KEPSC and 1.68% in KEPS) confirm that the EPS are acidic. Gas chromatography-mass spectrometry analyses demonstrated that the EPS consisted of rhamnose (29.83%), glucose (11.21%), galactose (52.45%), and mannose (6.50%). The treatment of an experimental pleurisy model in rats through subcutaneous administration of 50, 100, 200, and 400 mg/kg of KEPS decreased both the volume of inflammatory exudate and the number of leukocytes recruited to the pleural cavity. The present data showed that EPS production by K. oxytoca using the method described is easy to perform and results in a good yield. In addition, we show that KEPS exhibit anti-inflammatory activity when administered subcutaneously in rats.

Role of extracellular DNA in Enterococcus faecalis biofilm formation and its susceptibility to sodium hypochlorite

Abstract Objective This study investigated the role of extracellular deoxyribonucleic acid (eDNA) on Enterococcus faecalis ( E. faecalis ) biofilm and the susceptibility of E. faecalis to sodium hypochlorite (NaOCl). Methodology E. faecalis biofilm was formed in bovine tooth specimens and the biofilm was cultured with or without deoxyribonuclease (DNase), an inhibitor of eDNA. Then, the role of eDNA in E. faecalis growth and biofilm formation was investigated using colony forming unit (CFUs) counting, eDNA level assay, crystal violet staining, confocal laser scanning microscopy, and scanning electron microscopy. The susceptibility of E. faecalis biofilm to low (0.5%) or high (5%) NaOCl concentrations was also analyzed by CFU counting. Results CFUs and biofilm formation decreased significantly with DNase treatment (p<0.05). The microstructure of DNase-treated biofilms exhibited less structured features when compared to the control. The volume of exopolysaccharides in the DNase-treated biofilm was significantly lower than that of control (p<0.05). Moreover, the CFUs, eDNA level, biofilm formation, and exopolysaccharides volume were lower when the biofilm was treated with DNase de novo when compared to when DNase was applied to matured biofilm (p<0.05). E. faecalis in the biofilm was more susceptible to NaOCl when it was cultured with DNase (p<0.05). Furthermore, 0.5% NaOCl combined with DNase treatment was as efficient as 5% NaOCl alone regarding susceptibility (p>0.05). Conclusions Inhibition of eDNA leads to decrease of E. faecalis biofilm formation and increase of susceptibility of E. faecalis to NaOCl even at low concentrations. Therefore, our results suggest that inhibition of eDNA would be beneficial in facilitating the efficacy of NaOCl and reducing its concentration.

Exopolysaccharide production from Bacillus velezensis KY471306 using statistical experimental design

Abstract Exopolysaccharide (EPS) biopolymers produced by microorganisms play a crucial role in the environment such as health and bio-nanotechnology sectors, gelling agents in food and cosmetic industries in addition to bio-flocculants in the environmental sector as they are degradable, nontoxic. This study focuses on the improvement of EPS production through manipulation of different culture and environmental conditions using response surface methodology (RSM). Plackett-Burman design indicated that; molasses, yeast extract and incubation temperature are the most effective parameters. Box-Behnken RSM indicated that; the optimum concentration for each parameter was 12% (w/v) for molasses, 6 g/L yeast extract and 30 °C for incubation temperature. The most potent bacterial isolate was identified as Bacillus velezensis KY498625. After production, EPS was extracted, purified using DEAE-cellulose, identified using Fourier transform infrared (FTIR), gel permeation chromatography (GPC) and gas chromatography-mass spectroscopy (GC-MS). The result indicated that; it has molecular weight 1.14 × 105 D consisting of glucose, mannose and galactose.

Genotypic and phenotypic detection of capsular polysaccharides in Staphylococcus aureus isolated from bovine intramammary infections in Argentina

Staphylococcus aureus (n=157) isolated from intramammary infections in Argentine dairy areas were evaluated for presence of cap5 and cap8 loci. Isolates carrying cap5 and cap8 were serotyped using specific antisera. Sixty four percent of the isolates were genotyped as cap5 or cap8 and 50% of them expressed CP5 or 8.

Interactions of bacterial polysaccharides with cationic dyes: Physicochemical studies.

Capsular polysaccharides (SPS) are an integral component of gram-negative bacteria, and also have potential use as vaccine. In this paper, interactions of SPS isolated from Klebsiella strains K20 and K51 with cationic dyes pinacyanol chloride (PCYN) and acridine orange (AO) were studied by absorbance and fluorescence measurements. Both the polysaccharides having glucuronic acid as the potential anionic site induced strong metachromasy (blue shift ~100 nm) in the PCYN. The spectral changes were studied at different polymer/dye molar ratios (P/D = 0-40). A complete reversal of metachromasy was observed upon addition of co-solvents, suggesting the breakaway of dye molecules from the biopolymer matrix. Binding constant, changes in free energy, enthalpy and entropy of the dye polymer complex were also computed from the spectral data at different temperatures to reveal the nature of the interaction. Quenching of fluorescence of AO by the polymers and the incorporated mechanisms were also explored.

Fermentation technologies for the production of exopolysaccharide-synthesizing Lactobacillus rhamnosus concentrated cultures

The exopolysaccharide (EPS)-producing cultures such as Lactobacillus rhamnosus RW-9595M present a challenge for the culture producers because the high viscosity of the fermented growth medium makes it difficult to recover the cells by centrifugation or filtration. This study examined four approaches to reduce viscosity of the medium while producing high cell densities: incubation temperature, extended incubation in the stationary growth phase, production in alginate gel beads and fed-batch fermentation technology. Automated spectrophotometry (AS) was used to study the effects of temperature, pH and lactate level on growth of the strain. In AS assays, there was no significant difference in final maximal biomass production at temperatures ranging between 34 ºC to 44 ºC, but lower yields were noted at 46 C. A pH below 6.0 and a lactate concentration higher than 4 percent almost completely prevented growth. Under batch fermentation conditions, the viscosity of the medium obtained at 37 C was two fold higher than for 44 ºC. For cultures produced at 37 ºC, centrifugation at 10000 g during 5 min did not allow complete recovery of cells, in contrast to cultures grown at 44 ºC. An extended period of incubation (5 hrs) in the stationary growth phase did not reduce the final viscosity of the growth medium. For similar biomass levels, the glucose-based fed-batch fermentation allowed a 40 percent reduction in viscosity of the fermented medium in comparison to traditional batch cultures. High-density cell populations (3 x 10(10) CFU/g) were obtained when L. rhamnosus RW-9595M was grown in alginate beads. However, overall biomass yields in the immobilized cell bioreactor were half of those obtained in free-cell fermentations. Therefore three methods of producing concentrated EPS-producing cultures are proposed.

Polysaccharide production in batch process of Neisseria meningitidis serogroup C comparing Frantz, modified Frantz and Cartlin 6 cultivation media

Polysaccharide of N. meningitidis serogroup C constitutes the antigen for the vaccine against meningitis. The goal of this work was to compare three cultivation media for production of this polysaccharide: Frantz, modified Frantz medium (with replacement of glucose by glycerol), and Catlin 6 (a synthetic medium with glucose). The comparative criteria were based on the final polysaccharide concentrations and the yield coefficient cell/polysaccharide (Y P/X). The kinetic parameters: pH, substrate consumption and cell growth were also determined. For this purpose, 9 cultivation runs were carried out in a 80 L New Brunswick bioreactor, under the following conditions: 42 L of culture medium, temperature 35°C, air flow 5 L/min, agitation frequency 120 rpm and vessel pressure 6 psi, without dissolved oxygen or pH controls. The cultivation runs were divided in three groups, with 3 repetitions each. The cultivation using the Frantz medium presented the best results: average of final polysaccharide concentration = 0.134 g/L and Y P/X=0.121, followed by Catlin 6 medium, with results of 0.095 g/L and 0.067 respectively. Considering the principal advantages in the use of the synthetic medium, i.e. facilitation of a cultivation and purification steps of the polysaccharide production process, there is a possibility that in the near future, Catlin 6 will replace the traditional Frantz medium.